1. Field of the Invention:
This invention relates to amylase assays and to a reagent test kit for use in such assays. More particularly, it relates to amylase assays in which a polysaccharide is used as the amylase substrate.
2. Discussion of the Prior Art:
.alpha.-Amylase is an enzyme which hydrolyzes the .alpha.[1.fwdarw.4] linkages between the glucose units in starch and the lower polymers and oligomers of glucose. This enzyme is produced in the human body, primarily in the pancreas and in the salivary glands, and its concentration in various body fluids is a useful diagnostic tool for physicians. For example, in healthy individuals, serum .alpha.-amylase levels are relatively constant, but they rise in response to pathological conditions, such as acute pancreatitis.
U.S. Pat. No. 3,879,263, issued Apr. 22, 1975, and U.S. Pat. No. 4,000,042, issued Dec. 28, 1976, disclose a process and reagent test kit for use in determining the .alpha.-amylase content of a sample using the defined oligosaccharides maltotetraose, maltopentaose or maltohexaose as the amylase substrate. The reaction between .alpha.-amylase and these substrates, preferably in the presence of a maltase, produces a specific amount of glucose which can be measured by any conventional glucose detection system. The additional glucose detection step is an inconvenience. Furthermore, if glucose is present in the sample, it must either be removed or compensated for. Although this can be done by conventional techniques, it is an extra step in the process which is a disadvantage.
A. P. Jansen and P. G. A. B. Wydeveld, Nature, 182, 525 (1958) postulate that .alpha.-(p-nitrophenyl)maltoside could be a substrate for an amylase assay. However, this paper shows that the authors never identified the active agent responsible for their observations. They reported: (1) Incubation of samples of human urine, saliva, duodenal contents and only incidentally serum with .alpha.-(p-nitrophenyl)maltoside at 37.degree. for 16 hours produces 4-nitrophenol, identified spectrophotometrically by mixing the hydrolyzate with 0.02 N sodium hydroxide. (2) The hydrolysis was inhibited by protein precipitants such as 10% trichloroacetic acid and 0.5 N silver nitrate. (3) The hydrolysis was pH-dependent, being most effective at pH 5.9-7.0. They state that this experiment could not include "the possible existence of an unidentified carbohydrase" causing the observed activity. .alpha.-(4-Nitrophenyl)maltoside is not believed to be useful for human amylase assay because the cleavage of this compound by .alpha.-amylase is extremely slow. In contrast to the teaching of Jansen et al., the method of this invention produces amylase analyses in one hour or less.